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Life (Basel)
2024 Aug 20;148:. doi: 10.3390/life14081034.
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TMEM9B Regulates Endosomal ClC-3 and ClC-4 Transporters.
Festa M
,
Coppola MA
,
Angeli E
,
Tettey-Matey A
,
Giusto A
,
Mazza I
,
Gatta E
,
Barbieri R
,
Picollo A
,
Gavazzo P
,
Pusch M
,
Picco C
,
Sbrana F
.
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The nine-member CLC gene family of Cl- chloride-transporting membrane proteins is divided into plasma membrane-localized Cl- channels and endo-/lysosomal Cl-/H+ antiporters. Accessory proteins have been identified for ClC-K and ClC-2 channels and for the lysosomal ClC-7, but not the other CLCs. Here, we identified TMEM9 Domain Family Member B (TMEM9B), a single-span type I transmembrane protein of unknown function, to strongly interact with the neuronal endosomal ClC-3 and ClC-4 transporters. Co-expression of TMEM9B with ClC-3 or ClC-4 dramatically reduced transporter activity in Xenopus oocytes and transfected HEK cells. For ClC-3, TMEM9B also induced a slow component in the kinetics of the activation time course, suggesting direct interaction. Currents mediated by ClC-7 were hardly affected by TMEM9B, and ClC-1 currents were only slightly reduced, demonstrating specific interaction with ClC-3 and ClC-4. We obtained strong evidence for direct interaction by detecting significant Förster Resonance Energy Transfer (FRET), exploiting fluorescence lifetime microscopy-based (FLIM-FRET) techniques between TMEM9B and ClC-3 and ClC-4, but hardly any FRET with ClC-1 or ClC-7. The discovery of TMEM9B as a novel interaction partner of ClC-3 and ClC-4 might have important implications for the physiological role of these transporters in neuronal endosomal homeostasis and for a better understanding of the pathological mechanisms in CLCN3- and CLCN4-related pathological conditions.
GMR22T1029 Telethon Foundation, GJC22008 Telethon / Cariplo, Italy, ECS00000035 European Union-NextGenerationEU, Pilot grant CureCLCN4 charity, D34 Health Ministero dell'Università e della Ricerca
Figure 1. TMEM9B as a putative interactor of ClC-3 and ClC-4. (A). Network of ClC-3 interactors, among which is TMEM9B, from the BioGrid 4.4 database [28]. The green circle highlights the TMEM9B entry. (B). Network of ClC-4 interactors, among which there is TMEM9B. (C). Network of TMEM9B interactors, among which are ClC-3, -4, and -5, highlighted by green circles. (D). TMEM9B hydrophobicity plot showing a hydrophobic signal peptide (sequence positions 1–32) and a glycosylated asparagine at sequence position 60 in the extracellular/luminal domain. (E). TMEM9B AlphaFold predicted structure, highlighting, in cyan, the hydrophobic region from sequence positions 99 to 144, and, in magenta, the glycosylated asparagine at position 60. The signal peptide was removed from the AlphaFold structure and the image was created with PyMol.
Figure 2. Co-expression of ClC-4 and ClC-3 with TMEM9B in Xenopus oocytes. (A). Representative recordings of non-injected oocytes and oocytes injected with ClC-4 and with ClC-4 + TMEM9B evoked by the voltage-clamp protocol are shown on the right. (B). Averaged normalized I-V relationships of ClC-4 with and without TMEM9B. Currents are normalized as described in Methods. (C). Typical voltage clamp current traces of non-injected oocytes and oocytes injected with ClC-3 and co-injected with ClC-3 + TMEM9B in response to the stimulation protocol shown on the right. (D). Averaged normalized I-V currents collected for ClC-3 compared with ClC-3 co-injected with TMEM9B. Note that average currents from non-injected oocytes from the same batches are subtracted in the I-V plots and that, for some data points, error bars are smaller than symbol size.
Figure 3. Co-expression of ClC-1 with TMEM9B in Xenopus oocytes. Typical current traces recorded from oocytes injected with ClC-1 alone and with TMEM9B (A). Stimulation protocol is shown as inset. (B) shows the normalized conductance of ClC-1 compared with ClC-1 with TMEM9B. For ClC-1, the slope conductance is the most robust parameter to quantify functional expression [20]. The error bar indicates SD (n = 3 injections). The star indicates p < 0.05 (Student’s t-test).
Figure 4. Co-expression with TMEM9B strongly reduced transport currents of ClC-4 in HEK cells. (A). Representative ionic currents elicited by the voltage-clamp protocol shown in the inset in control conditions (left trace) and in the presence of TMEM9B (right trace). (B). Average I-V plot shows a strong reduction of outward ClC-4 currents by TMEM9B (mean ± SEM). (C). Average current values at 200 mV (mean ± SD) (red bar, n = 11, I(200 mV) = 1.15 ± 0.45 nA; blue bar, n = 10, I(200 mV) = 0.14 ± 0.07 nA, p < 0.0001).
Figure 5. TMEM9B modulates the biophysical properties of ClC-3. (A). Representative ClC-3 currents elicited by the voltage-clamp protocol shown in the inset in control conditions (left trace) and in the presence of TMEM9B (right trace).0 (B). Representative recordings from a ClC-3 transfected cell (left trace) and from a cell co-transfected with TMEM9B using long (500 ms) pulses as indicated in the inset. (C). Average I-V plot in the absence (orange) and presence of TMEM9B (light blue, mean ± SEM). (D). Average current values at 200 mV (mean ± SD, orange bar, n = 13, I(200 mV) = 2.06 ± 0.97 nA; light blue bar, n = 13, I(200 mV) = 0.64 ± 0.37 nA; The four stars indicate p = 0.0002 (Student’s t-test). (E). Slowing of current activation by TMEM9B. Activation kinetics of cells co-transfected with TMEM9B was fitted with a double exponential function and values of the extracted time constants are shown as mean ± SD (purple bar: τfast =21.0 ± 6.4 ms; pink bar: τslow =120 ± 75 ms). No such slow kinetics were seen in cells transfected only with ClC-3.
Figure 6. Co-expression with TMEM9B does not affect ClC-7 transport currents in HEK cells. (A). Representative ClC-7 currents elicited by the voltage-clamp protocol shown in the inset in control conditions (left trace) and in a cell co-transfected with TMEM9B (right trace). (B). Average I-V plot of ClC-7 transfected cells in the absence (green) and presence of TMEM9B (purple, mean ± SEM). (C). Average current values at 140 mV (mean ± SD, green bar, n = 5, I(200 mV) = 1.38 ± 0.58 nA; purple bar, n = 4, I(200 mV) = 1.19 ± 0.59 nA, p = 0.649).
Figure 7. Subcellular localization of TMEM9B expressed alone. (A). Confocal images of HEK cells transfected with TMEM9B-GFP (green), stained with CellMask_DeepRed (magenta), merged image, and corresponding bright field image. The squared region is shown zoomed on the right. (B). Confocal images of cells transfected with TMEM9B-GFP (green) and stained with Lysotracker_DeepRed (magenta), merged image, and corresponding bright field image. The squared region is shown zoomed on the right. (C). Similar to B, but using mCherry tagged TMEM9B (red).
Figure 8. Subcellular localization of TMEM9B expressed with CLC proteins. (A). Confocal images of HEK cells co-transfected with TMEM9B-mCherry (red) and ClC-4-GFP (green), stained with CellMask_DeepRed (cyan), the merged image, and the corresponding bright field image. The squared region is shown zoomed on the right. (B). Similar results with inverted tags, i.e., TMEM9B-GFP (green) and ClC-4-mCherry (red). (C). Similar results for cells co-transfected with TMEM9B-mCherry (red) and ClC-3-GFP (green). (D). Similar results for cells co-transfected with TMEM9B-mCherry (red) and ClC-1-GFP (green). (E). Similar results for cells co-transfected with TMEM9B-mCherry (red) and ClC-7-GFP (green).
Figure 9. FLIM-FRET analysis of TMEM9B co-expressed with CLC proteins. (A). ClC-4-GFP phasor plot with corresponding lifetime value and representative image of a ClC-4-GFP transfected HEK cell. (B). ClC-4-GFP/TMEM9B-mCherry phasor plot with relative lifetime value and representative fluorescent confocal merged image of a ClC-4-GFP/TMEM9B-mCherry co-transfected HEK cell. Here, and in panels D, F, and H, the purple circle indicates the GS-coordinates of the unquenched donor. (C). ClC-3-GFP phasor plot with relative lifetime value and representative fluorescent confocal merged image of a ClC-3-GFP transfected HEK cell. (D). ClC-3-GFP/TMEM9B-mCherry phasor plot with relative lifetime value and representative fluorescent confocal merged image of a ClC-3-GFP/TMEM9B-mCherry co-transfected HEK cell. (E). ClC-1-GFP phasor plot with relative lifetime value and representative image of a ClC-1-GFP transfected HEK cell. (F). ClC-1-GFP/TMEM9B-mCherry phasor plot with relative lifetime value and representative image of a ClC-1-GFP/TMEM9B-mCherry co-transfected HEK cell. (G). ClC-7-GFP phasor plot with relative lifetime value and representative image of a ClC-7-GFP transfected HEK cell. (H). ClC-7-GFP/TMEM9B-mCherry phasor plot with relative lifetime value and representative image of a ClC-7-GFP/TMEM9B-mCherry co-transfected HEK cell. (I). FRET Efficiency analysis comparison with **** p < 0.0001 compared to the other groups.
